2008 Rustbelt RNA Meeting
RRM

 

Registration

Agenda

Abstracts

Directions

Poster abstracts

Poster number 46 submitted by David Mess

Probing catalytic activity of tRNAHis-guanylyltransferase using small RNA substrates

David S. Mess (Biochemistry, Ohio State University), Jane E. Jackman (Biochemistry, Ohio State University)

Abstract:
All tRNAHis contain an additional G residue (G-1) that is necessary for aminoacylation by the cognate synthetase (HisRS). G-1 is added post-transcriptionally in eukaryotes, across from a universally conserved A at position 73, by an essential enzyme, the tRNAHis guanylyltransferase (Thg1).
The known physiological substrate for Thg1, tRNAHis, shares the highly conserved structure of all tRNAs, yet must be individually recognized by Thg1 for selective G-1 addition; the molecular basis of this recognition is not entirely understood. Insight into tRNA recognition by HisRS has been achieved using small RNA stem-loop substrates. Consequently, we measured steady-state kinetic parameters for Thg1 activity with two small RNA stem-loops. Following nomenclature used for HisRS, the first substrate (micro-His) mimics the 7 bp acceptor stem of tRNAHis, and the second substrate (mini-His) mimics the acceptor stem and 5 bp T stem, which coaxially stack in full-length (FL) tRNA. Thg1 exhibits nearly identical kcat values for G-1 addition with micro-His and mini-His. The measured Km’s for these substrates reveal an intriguing trend since Km for mini-His is ~5-fold greater than Km for FL tRNAHis, yet Km does not continue to increase as the substrate is shortened further. Instead Km for micro-His is surprisingly low, similar to that measured for FL tRNA.
Thg1 catalyzes a second biochemical reaction, 3’-5’ addition of multiple nucleotides to tRNA substrates where A73 has been substituted with C. Therefore, we made analogous substitutions in the small RNAs and found that for both substrates, substitution of C resulted in ~30-fold increase in kcat/Km compared to its A-containing counterpart, mainly driven by an increased kcat. Since these substrates are otherwise identical, these data suggest that increased catalytic activity is attributable to an enhanced rate under conditions where Watson-Crick base pairs are formed. Since kcat/Km for C-containing micro-His is within 2-fold of that observed for wild-type FL tRNAHis, non-tRNA species could be catalytically competent enough to act as physiologically relevant Thg1 substrates in vivo.

Keywords: Thg1, tRNAHis, catalysis